Downregulation of the KIP family members p27KIP1 and p57KIP2 by SKP2 and the role of methylation in p57KIP2 inactivation in nonsmall cell lung cancer

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Abstract
Knowing the status of molecules involved in cell cycle control in cancer is vital for therapeutic approaches aiming at their restoration. The p27KIP1 and p57KIP2 cyclin‐dependent kinase inhibitors are nodal factors controlling normal cell cycle. Their expression in normal lung raises the question whether they have a mutual exclusive or redundant role in nonsmall cell lung cancer (NSCLC). A comparative comprehensive analysis was performed in a series of 70 NSCLCs. The majority of cases showed significantly reduced expression of both members compared to normal counterparts. Low KIP protein levels correlated with increased proliferation, which seems to be histological subtype preponderant. At mechanistic level, degradation by SKP2 was demonstrated, in vivo and in vitro, by siRNA‐methodology, to be the most important downregulating mechanism of both KIPs in NSCLC. Decreased p57KIP2‐transcription complements the above procedure in lowering p57KIP2‐protein levels. Methylation was the main cause of decreased p57KIP2‐mRNA levels. Allelic loss and imprinting from LIT1 mRNA contribute also to decreased p57KIP2 transcription. In vitro recapitulation of the in vivo findings, in A549 lung cells (INK4A‐B(−/−)), suggested that inhibition of the SKP2‐degradation mechanism restores p27KIP1 and p57KIP2 expression. Double siRNA treatments demonstrated that each KIP is independently capable of restraining cell growth. An additional demethylation step is required for complete reconstitution of p57KIP2 expression in NSCLC.